Vaccination is one of the greatest public health success stories. Nowadays vaccines that protect against many of the viruses that once caused fatal childhood diseases are routinely used throughout the world. However, traditional methods of vaccine development using inactivation or attenuation of viruses have failed for some of the most deadly human pathogens, necessitating new approaches. Advances in molecular virology have enabled the genetic manipulation of viruses, which has opened new opportunities for vaccine development. Genetic modification of viruses not only allows for their attenuation but also for incorporation of sequences from other viruses, turning one pathogen into a vaccine carrier for another. Viral vectors have been studied as potential tools to deliver vaccines as they present advantages over traditional vaccines in that they stimulate a broad range of immune responses including antibody (B cell), T helper cell (CD4+ T cell), and cytotoxic T lymphocyte (CTL, CD8+ T cell) mediated immunity. These viral vector vaccines could be used against various infectious and malignant diseases (Polo and Dubensky, Jr. Drug Discov Today. 2002, Jul. 1; 7(13):719-27; Small and Hildegund, Curr Opin Virol. 2011, Oct. 1; 1(4): 241-245). However, there are limitations on the use of viral vector-based vaccines. For example, preexisting anti-vector immunity against the viral vectors that potentially inactivate the vaccine presents an issue as does the limited cloning capacity for the transgene of interest. Numerous vaccine investigations are in progress to improve the efficiency of this technology and to overcome its limitations (Nayak and Herzog. Gene Therapy, 2009, 17 (3): 295-304).
Enveloped RNA viruses have highly organized structures. One or more nucleocapsid proteins encapsidate their RNA, matrix proteins often lie between the capsid and the membrane, and one or more transmembrane glycoproteins interact with the matrix or nucleocapsid proteins to direct efficient particle assembly. Once the particles are released from cells, one or more glycoproteins bind cellular receptors and catalyze membrane fusion to allow the viruses to enter new cells.
Alphaviruses such as Semliki Forest Virus (SFV) are positive-strand, membrane-enveloped RNA viruses that encode four non-structural proteins called nsP 1-4 and three structural proteins: capsid, and the E1 and E2 transmembrane glycoproteins. The nsp 1-4 proteins are translated from the first two-thirds of the genomic RNA. These proteins form a complex which directs replication of the genomic RNA to form antigenomic RNA, which is then copied to form full-length positive strand RNA and a subgenomic mRNA that encodes the structural proteins. The capsid protein encases the genomic RNA in the cytoplasm to generate a nucleocapsid that buds from the cell surface in a membrane containing the SFV glycoproteins. Alphavirus RNA replication occurs inside light-bulb shaped, membrane-bound compartments called spherules that initially form on the cell surface and are then endocytosed to form cytopathic vacuoles containing multiple spherules (Spuul et al., 2010. Journal of virology 84:7543-7557). The replicase proteins are localized to the cytoplasmic side of the spherules (Froshauer et al., 1988. The Journal of cell biology 107:2075-2086). How the replicated RNA in the spherules is packaged into nucleocapsids prior to SFV budding is not known. Alphavirus RNA replicons lacking any structural protein genes can still replicate efficiently inside a cell, but they are incapable of propagating beyond the cell.
Vesicular stomatitis virus (VSV) is a negative-strand RNA virus that encodes a single membrane glycoprotein (G), a matrix protein, and a nucleocapsid protein as well as two proteins that form the viral polymerase (Rose and Whitt, 2001. Rhabdoviridae: The Viruses and Their Replication, p. 1221-1240. In D. Knipe and P. Howley (ed.), Fields' Virology. Lippencott-Raven, Philadelphia). Remarkably, when cells are transfected with an SFV RNA replicon encoding only the SFV non-structural proteins and the VSV G protein, infectious membrane-enveloped vesicles containing the VSV G protein are generated (Rolls et al., 1994. Cell 79:497-506). These infectious, virus-like vesicles (VLVs) grow to low titers, but can be passaged like a virus in tissue culture cells. The particles contain the replicon RNA and VSV G protein, but unlike enveloped RNA viruses, have a very low density because they do not contain a tightly-packed nucleocapsid protein around the RNA (Rolls et al., 1994. Cell 79:497-506). The mechanism by which these VLVs are generated has not been determined. These VLVs expressing other proteins have proven useful as experimental vaccines (Rose et al., 2008. PNAS 105:5839-5843; Schell et al., 2011. Journal of virology 85:5764-5772). But, there are two significant limitations of the VLV system as a vaccine platform: the relatively low titer of the VLVs and rapid loss of expression of the foreign genes upon passage.
Clearly, there is a need in the art for methods of producing more efficient virus-vector vaccine systems that support stable foreign gene expression, while generating high vaccine titers that induce a potent immune response. The present invention fulfills this need.